PARP1 trapping activates cGAS-STING pathway to induce immunogenic cell death in multiple myeloma

Alternative Non-Homologous End Joining (Alt-NHEJ) DNA repair is considered a major player in cancer genomic instability. Here, we investigated cGAS-STING pathway as crucial node in the interplay between Alt-NHEJ repair and immune response, in the aim to discover novel therapeutic vulnerability in Multiple Myeloma (MM).  METHODS: In silico analyses were performed by querying publicly available MM datasets (GSE66293 and CoMMpass). Anti-proliferative activity was evaluated by CellTiter-Glo, while flow cytometry analysis was used to determine the apoptotic process, cell cycle, phagocytosis, micronuclei detection, Calreticulin and T-cell activation markers. Protein expression was detected by western blot of whole or fractioned protein extracts.

By interrogating public MM datasets, a significant correlation between hyperactivation of cGAS-STING mRNA signature and poor PFS and OS in MM was observed. Indeed, Gene Set Enrichment Analysis (GSEA) showed enrichment of DNA repair, TNFA signaling and oxidative phosphorylation in patients with cGAS-STING activation patients, associated to higher mRNA expression of DNA Ligase 3 (LIG3) and PARP1. On this basis, we evaluated the activity of Alt-NHEJ inhibitor Talazoparib (PARP1-inhibitor) on MM cell lines, focusing on their capability to modulate cGAS-STING pathway. We first detected a significant reduction of cell proliferation and the induction of apoptosis following Talazoparib treatment, which in turn induced DNA damage response and cell cycle blockade, and finally cGAS-STING pathway activation as result of PARP1-trapping into chromatin. Next, by performing co-culture experiments with healthy donor's peripheral blood mononuclear cells (PBMCs), we finally demonstrated the induction of immunogenic cell death, which was abrogated in cGAS-knockout cells, underscoring the pathway's functional relevance.

Taken together, our findings indicate that Alt-NHEJ inhibitors are potential immune-stimulating agents for MM with hyperactivation of cGAS-STING pathway, coherently with our working hypothesis.